Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Insects of the Order Coleoptera (coleopterans) are an important group of agricultural pests which cause extensive damage to crops each year. There are a number of beetles that cause significant economic damage; examples include Chrysomelid beetles (such as flea beetles and corn rootworms) and Curculionids (such as alfalfa weevils).
Flea beetles include a large number of genera (e.g., Altica, Apphthona, Argopistes, Disonycha, Epitrix, Longitarsus, Prodagricomela, Systena, Psylliodes, and Phyllotreta). Phyllotreta striolata includes the striped flea beetle. Phyllotreta cruciferae includes the canola flea beetle, the rape flea beetle, and the crucifer flea beetle. Canola, also known as rape, is an oil seed brassica (e.g., Brassica campestris, Brassica rapa, Brassica napus, and Brassica juncea).
Flea beetles include a large number of beetles that feed on the leaves of a number of grasses, cereals, and herbs. Phyllotreta cruciferae, Phyllotreta striolata, and Phyllotreta undulata, are particularly destructive annual pests that attack the leaves, stems, pods, and root tissues of susceptible plants. Psylliodes chrysocephala, a flea beetle, is also a destructive, biennial pest that attacks the stems and leaves of susceptible plants.
Chemical pesticides have provided effective pest control; however, the public has become concerned about contamination of food with residual chemicals and of the environment, including soil, surface water, and ground water. Working with pesticides may also pose hazards to the persons applying them. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides form the marketplace could limit economical and effective options for controlling costly pests.
In addition, the regular use of pesticides for the control of unwanted organisms can select for resistant strains. This has occurred in many species of economically important insects and other pests. The development of pesticide resistance necessitates a continuing search for new control agents having different modes of action.
Thus, there is an urgent need to identify new methods and compositions for controlling pests, such as the many different types of coleopterans that cause considerable damage to susceptible plants.
Certain strains of the soil microbe Bacillus thuringiensis (B.t.), a Gram-positive, spore-forming bacterinum, can be characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and are specific in their toxic activity. These xcex4-endotoxins, which are produced by certain B.t. strains, are synthesized by sporulating cells. Certain types of B.t. toxins, upon being ingested by a susceptible insect, are transformed into biologically active moieties by the insect gut juice proteases. The primary target is cells of the insect gut epithelium, which are rapidly destroyed by the toxin.
Certain Bacillus toxin genes have been isolated and sequenced. The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature. In addition, with the use of genetic engineering techniques, new approaches for delivering these Bacillus toxins to agricultural environments are under development, including the use of plants genetically engineered with toxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles. Recombinant DNA-based B.t. products have been produced and approved for use. Thus, isolated Bacillus toxin genes are becoming commercially valuable.
Until fairly recently, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran(caterpillar)pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline xcex4-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, new subspecies of B.t. have been identified, and investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively.
Hxc3x6fte and Whiteley (Hxc3x6fte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255) classified B.t. crystal protein genes into four major classes. The classes were Cryl (Lepidoptera-specific),CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). CryV and CryVI were proposed to designate a class of toxin genes that are nematode-specific. Other classes of B.t. genes have now been identified.
The 1989 nomenclature and classification scheme of Hxc3x6fte and Whiteley for crystal proteins was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. As more toxin genes were discovered, that system started to become unworkable, as genes with similar sequences were found to have significantly different insecticidal specificities. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmore et al. [1996] Society for Invertebrate Pathology, 29th Annual Meeting, 3rd International Colloquium on Bacillus thuringiensis, University of Cordoba, Cordoba, Spain, September 1-6, abstract). The mnemonic xe2x80x9ccryxe2x80x9d has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, with the noted exceptions, although a number have been reclassified. See also xe2x80x9cRevisions of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins,xe2x80x9d N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean, Microbiology and Molecular Biology Reviews (1998) Vol. 62:807-813; and Crickmore, Zeigler, Feitelson, Schnepf, Van Rie, Lereclus, Baum, and Dean, xe2x80x9cBacillus thuringiensis toxin nomenclaturexe2x80x9d (1999) http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html. That system uses the freely available software applications CLUSTAL W and PHYLIP. The NEIGHBOR application within the PHYLIP package uses an arithmetic averages (UPGMA) algorithm.
B.t. isolate PS86A1 is disclosed in the following U.S. Pat. No. 4,849,217 (activity against alfalfa weevil); U.S. Pat. No. 5,208,017 (activity against corn rootworm); U.S. Pat. No. 5,286,485 (activity against lepidopterans); and U.S. Pat. No. 5,427,786 (activity against Phyllotreta genera). A gene from PS86A1 was cloned into B.t. MR506, which is disclosed in U.S. Pat. No. 5,670,365 (activity against nematodes) and PCT international patent application publication No. WO93/04587 (activity against lepidopterans). The sequences of a gene and a Cry6A (CryVIA) toxin from PS86A1 are disclosed in the following U.S. Pat. No. 5,186,934 (activity against Hypera genera); U.S. Pat. No. 5,273,746 (lice); U.S. Pat. Nos. 5,262,158 and 5,424,410 (activity against mites); as well as in PCT international patent application publication No. WO94/23036 (activity against wireworms). U.S. Pat. Nos. 5,262,159 and 5,468,636, disclose PS86A1, the sequence of a gene and toxin therefrom, and a generic formula for toxins having activity against aphids.
B.t. isolate PS52A1 is disclosed by the following U.S. patents as being active against nematodes: U.S. Pat. Nos. 4,861,595; 4,948,734, 5,093,120, 5,262,399, 5,236,843, 5,322,932; and 5,670,365. PS52A1 is also disclosed in U.S. Pat. No. 4,849,217,supra, and PCT international patent application publication No. WO95/02694 (activity against Calliphoridae). The sequences of a gene and a nematode-active toxin from PS52A1 are disclosed in U.S. Pat. No. 5,439,881 and European patent application publication No. EP 0462721. PS52A1, the sequence of a gene and nematode-activetoxin therefrom, and a generic formula for CryVIA toxins are disclosed in PCT international patent application publication No. WO 92/19739.
As a result of extensive research, other patents have issued for new B.t. isolates and new uses of B.t. isolates. However, the discovery of new Bacillus isolates, toxins, and genes, and new uses of known B.t. isolates remains an empirical, unpredictable art.
Although B.t. strains PS86A1 and PS52A1, and a gene and toxin therefrom, were known to have certain pesticidal activity, additional genes encoding active toxins from these isolates were not previously known in the art.
The subject invention provides novel genes encoding pesticidal toxins. Preferred, novel toxin genes of the subject invention are designated 86A1(b) and 52A1(b). These genes encode toxins that are active against plant pests, preferably insects, preferably coleopterans, and most preferably flea beetles of the genus Phyllotreta.
In a preferred embodiment, the subject invention concerns plants and plant cells transformed with at least one polynucleotide sequence of the subject invention such that the transformed plant cells express pesticidal toxins in tissues consumed by the target pests. Plants are transformed in this manner in order to confer pest resistance upon said plants. In these preferred embodiments, pests contact the toxins expressed by the transformed plant by ingesting or consuming the plant tissues expressingthe toxin. Such transformation of plants can be accomplishedusing techniques known to those skilled in the art. Proteins expressed in this manner are better protected from environmental degradation and inactivation. There are numerous other benefits of using transformed plants of the subject invention.
In an alternative embodiment, B.t. isolates of the subject invention, or recombinant microbes expressing the toxins described herein, can be used to control pests. Thus, the subject invention includes substantially intact B.t. cells, and recombinant cells containing the expressed toxins of the invention, treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes active upon ingestion by a target insect.
Another aspect of the subject invention includes synthetic, plant-optimized B.t. genes that are particularly well suited for providing stable maintenance and expression in the transformed plant.
SEQ ID NO. 1 is a forward oligonucleotide probe for 52A1(b) and 86A1(b).
SEQ ID NO. 2 is a nucleotide sequence of a gene encoding the 86A1 (b) toxin.
SEQ ID NO. 3 is an amino acid sequence of the 86A1 (b) toxin.
SEQ ID NO. 4 is a nucleotide sequence of a gene encoding the 52A1(b) toxin.
SEQ ID NO. 5 is an amino acid sequence of the 52A1(b) toxin.
SEQ ID NO. 6 is a nucleotide sequence of the plant-optimized MR510 gene.
SEQ ID NO. 7 is an amino acid sequence encoded by the plant-optimized MR 510 gene.
SEQ ID NO. 8 is a preferred, truncated version of the full-length, native 52A1(b) toxin. In the gene encoding this toxin (and for the genes encoding all of the following amino acid sequences shown in SEQ ID NOS. 9-19), the initiator codon for methionine has been added so that the N-terminal amino acid is methionine and not leucine (leucine is the first amino acid in the native protein). This truncation and the proteins shown in SEQ ID NOS. 9-13 have N-terminal deletions from the native protein. The natural 52A1(b) end is otherwise used in these truncations. After the first amino acid, this truncated toxin begins with amino acid 10 of the native protein. That is, the first 9 amino acids of the native protein have been replaced in favor of the single amino acid methionine. The remaining (C-terminal) portion of this toxin is the same as that of the native protein. In preferred embodiments, two stop codons are used in the gene encoding this toxin as well as in the genes encoding the following truncated proteins (SEQ ID NOS. 9-19).
SEQ ID NO. 9 is another preferred, truncated version of the full-length, native 52A1(b) protein. This protein comprises methionine added to the native protein beginning at amino acid 21 of the native protein. Thus, the first 20 N-terminal amino acids of the native protein have been replaced with methionine.
SEQ ID NO. 10 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncation, the first 26 N-terminal amino acids ofthe native protein have been replaced with methionine.
SEQ ID NO. 11 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncation, the first 41 N-terminal amino acids of the native protein have been replaced with methionine.
SEQ ID NO. 12 is another preferred, truncated version of the full-length, native 52A1 (b) protein. In this truncation, the first 52 N-terminal amino acids of the native protein have been replaced with methionine.
SEQ ID NO. 13 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncation, the first 74 N-terminal amino acids ofthe native protein have been replaced with methionine.
SEQ ID NO. 14 is another preferred, truncated version of the full-length, native 52A1 (b) protein. In this truncation (and in the remaining truncations shown in SEQ ID NOS. 15-19), the natural beginning of the 52A1(b) protein (with the exception that leucine has been replaced with methionine) is used. Thus, these toxins (and the genes encoding them) are the result of making C-terminal deletions to the native protein. In this truncated protein, 93 amino acids are removed from the C-terminus of the native protein. Thus, this truncated protein ends with amino acid 269 of the native protein.
SEQ ID NO. 15 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncated protein, 82 amino acids are removed from the C-terminus of the native protein. Thus, this truncated protein ends with amino. acid 280 of the native protein.
SEQ ID NO. 16 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncated protein, 74 amino acids are removed from the C-terminus of the native protein. Thus, this truncated protein ends with amino acid 288 of the native protein.
SEQ ID NO. 17 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncatedprotein, 30 amino acids are removed from the C-terminus of the native protein. Thus, this truncated protein ends with amino acid 332 of the native protein.
SEQ ID NO. 18 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncated protein, 20 amino acids are removed from the C-terminus of the native protein. Thus, this truncated protein ends with amino acid 342 of the native protein.
SEQ ID NO. 19 is another preferred, truncated version of the full-length, native 52A1(b) protein. In this truncated protein, three amino acids are removed from the C-terminus of the native protein. Thus, this truncated protein ends with amino acid 359 of the native protein.
The subject invention provides novel genes encoding pesticidal toxins. Preferred, novel toxin genes of the subject invention are designated 86A1(b) and 52A1(b). These genes encode toxins that are active against (which can be used to control, or which are toxic to, or which are lethal to) plant pests, preferably insects, preferably coleopterans, and most preferably flea beetles of the genus Phyllotreta. The use of the subject genes and toxins for controlling other pests, such as pests of the genus Psylliodes, is also contemplated.
In a preferred embodiment, the subject invention concerns plants and plant cells transformed with at least one polynucleotide sequence of the subject invention such that the transformed plant cells express pesticidal toxins in tissues consumed by the target pests. Plants are transformed in this manner in order to confer pest resistance upon said plants. In these preferred embodiments, pests contact the toxins expressed by the transformed plant by ingesting or consuming the plant tissues expressing the toxin. Such transformation of plants can be accomplished using techniques known to those skilled in the art. Proteins expressed in this manner are better protected from environmental degradation and inactivation. There are numerous other benefits of using transformed plants of the subject invention.
In an alternative embodiment, B.t. isolates of the subject invention, or recombinant microbes expressing the toxins described herein, can be used to control pests. Thus, the subject invention includes substantially intact B.t. cells, and recombinant cells containing the expressed toxins of the invention. These cells can be treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest. See, e.g., U.S. Pat. Nos. 4,695,462; 4,861,595; and 4,695,455. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes active upon ingestion by a target insect.
Characteristics of Bacillus thuringiensis isolates PS86A1 and PS52A1, such as colony morphology, inclusiontype, and the sizes of alkali-solubleproteins (by SDS-PAGE), have been disclosed in, for example, U.S. Pat. No. 5,427,786 and published PCT application WO 95/02694, respectively.
Isolates useful according to the subject invention are available by virtue of deposits described in various U.S. patents. Examples of such patents are discussed in more detail in the Background section, supra. The cultures disclosed in this application have been deposited in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA.
The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of a deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
Genes and Toxins
Certain DNA sequences of the subject invention have been specifically exemplified herein. These sequences are exemplary of the subject invention. It should be readily apparent that the subject invention includes not only the genes and sequences specifically exemplified herein but also equivalents, variants, variations, mutants, fusions, chimerics, truncations, fragments, and smaller genes that exhibit the same or similar characteristics relating to pesticidal activity and expression in plants, as compared to those specifically disclosed herein.
Fragments of the genes and toxins specifically exemplified herein which retain the pesticidal activity of the exemplified toxins are within the scope of the subject invention. Genes and toxins useful according to the subject invention include not only the full length sequences but also fragments of these sequences which retain the characteristic pesticidal activity of the toxins specifically exemplified herein.
Variant DNA sequences are within the scope of the subject invention. As used herein, xe2x80x9cvariantsxe2x80x9d and xe2x80x9cequivalentsxe2x80x9d refer to sequences which have nucleotide (or amino acid) substitutions, deletions, additions, or insertions which do not materially affect the expression of the subject genes, and the resultant pesticidal activity of the encoded toxins, particularly in plants. As used herein, the terms xe2x80x9cvariantsxe2x80x9d or xe2x80x9cvariationsxe2x80x9d of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term xe2x80x9cequivalent toxinsxe2x80x9d refers to toxins having the same or essentially the same biological activity against the target pests as the exemplified toxins.
Genes can be modified, and variations of genes may be readily constructed, as would be known to one skilled in the art. For example, U.S. Pat. No. 5,605,793 describes methods for generating additional molecular diversity by using DNA reassembly after random fragmentation. Standard techniques are available for making point mutations. The use of site-directed mutagenesis is known in the art. Fragments of the subject genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or can be used to systematically cut off nucleotides from the ends of these genes. Useful genes may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.
Because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to xe2x80x9cessentially the samexe2x80x9d sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity.
It should be apparent to a person skilled in this art that, given the sequences of the genes and toxins as set forth herein, the genes and toxins of the subject invention can be obtained through several means. For example, the subject genes may be constructed synthetically by using a gene synthesizer. The subject genes and toxins can also be derived from wild-type genes and toxins from isolates deposited at a culture depository as described above. Equivalenttoxins and/or genes encoding these equivalenttoxins can be derived from Bacillus isolates and/or DNA libraries using the teachings provided herein.
As the skilled artisan would readily recognize, DNA can exist in a double-stranded form. In this arrangement, one strand is complementary to the other strand and vice versa The xe2x80x9ccoding strandxe2x80x9d is often used in the art to refer to the strand having a series of codons (a codon is three nucleotides that can be read three-at-a-time to yield a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest. In order to express a protein in vivo, a strand of DNA is typically translated into a complementary strand of RNA which is used as the template for the protein. As DNA is replicated in a plant (for example) additional, complementary strands of DNA are produced. Thus, the subject invention includes the use of either the exemplified polynucleotides shown in the attached sequence listing or the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exemplified DNA are included in the subject invention. Thus, in preferred embodiments, the direct or indirect expression of the subject polynucleotide results, directly or indirectly, in the intracellular production and maintenance of the desired polypeptide or protein.
There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other Bacillus toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins or fragments of these toxins, can readily be prepared using standard procedures in this art.
Certain toxins of the subject invention have been specifically exemplified herein; these toxins are merely exemplary of the toxins of the subject invention. It is readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent genes will encode toxins that have high amino acid identity or homology with the toxins coded for by the subject genes. Equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid identity will typically be greater than 60%, preferably be greater than 75%, more preferably greater than 80%, more preferably greaterthan 90%, and can be greaterthan 95%. These identities are as determined using standard alignment techniques. Preferred methods of determining percent identity are discussed in Crickmore et al., supra. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 2 provides a listing of examples of amino acids belonging to each class.
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutionsmust not significantly detract from the biological activity of the toxin.
As used herein, referenceto xe2x80x9cisolatedxe2x80x9d polynucleotidesand/orxe2x80x9cpurifiedxe2x80x9d toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature, and would include their use in plants. Thus, reference to xe2x80x9cisolated and purifiedxe2x80x9d signifies the involvement of the xe2x80x9chand of manxe2x80x9d as described herein. Chimeric toxins and genes also involve the xe2x80x9chand of man.xe2x80x9d
Full length B.t. toxins can be expressed and then converted to active, truncated forms through the addition of appropriate reagents and/or by growing the cultures under conditions which result in the truncation of the proteins through the fortuitous action of endogenous proteases. In an alternative embodiment, the full length toxin may undergo other modifications to yield the active form of the toxin. Adjustment of the solubilization of the toxin, as well as other reaction conditions, such as pH, ionic strength, or redox potential, can be used to effect the desired modification of the toxin. Truncated toxins of the subject invention can be obtained by treating the crystalline xcex4-endotoxin of Bacillus thuringiensis with a serine protease such as bovine trypsin at an alkaline pH and preferably in the absence of xcex2-mercaptoethanol.
Chimeric and/or fusion genes and toxins (typically produced by either combining portions from more than one Bacillus toxin or gene, or by combining full-length genes and toxins, and combinations thereof) may also be utilized according to the teachings of the subject invention. The subject invention includes the use of all or part of the toxins and genes in the production of fusion proteins and fusion genes. Chimeric toxins can also be produced by combining portions of multiple toxins.
Methods have been developed for making useful chimeric toxins by combining portions of B.t. crystal proteins. The portions which are combined need not, themselves, be pesticidal so long as the combination of portions creates a chimeric protein which is pesticidal. This can be done using restriction enzymes, as described in, for example, European Patent 0 228 838; Ge, A.Z., N. L. Shivarova, D. H. Dean (1989) Proc. Natl. Acad Sci. USA 86:4037-4041; Ge, A.Z., D. Rivers, R. Milne, D. H. Dean (1991) J. Biol. Chem. 266:17954-17958; Schnepf, H. E., K. Tomczak, J. P. Ortega, H. R. Whiteley (1990) J. Biol. Chem. 265:20923-20930; Honee, G., D. Convents, J. Van Rie, S. Jansens, M. Peferoen, B. Visser (1991) Mol. Microbiol. 5:2799-2806. Alternatively, recombination using cellular recombination mechanisms can be used to achieve similar results. See, for example, Caramori, T., A. M. Albertini, A. Galizzi (1991) Gene 98:37-44; Widner, W.R., H. R. Whiteley (1990) J. Bacteriol. 172:2826-2832; Bosch, D., B. Schipper, H. van der Kliej, R. A. de Maagd, W. J. Stickema (1994) Biotechnology 12:915-918. A number of other methods are known in the art by which such chimeric DNAs can be made. The subject invention is meant to include chimeric proteins that utilize the novel sequences identified in the subject application.
In addition, toxins of the subject invention may be used in combination with each other or with other toxins to achieve enhanced pest control. Of course, this includes the use of the subject toxins with different toxins in pest-control schemes designed to control pests that might have developed resistance against one or more toxins.
With the teachings provided herein, one skilled in the art could readily produce and use the various toxins and polynucleotide sequences described herein.
Recombinant Hosts and Other Application Methods
The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. As used herein, the term xe2x80x9cheterologousxe2x80x9d gene refers to a gene that does not naturally occur in the host that is transformed with the gene. In preferred embodiments, expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide.
When transformed plants of the subject invention are ingested by the pest, the pests will ingest the toxin. The result is a control of the pest. Benefits of in planta expression of the toxin proteins include improved protection of the pesticide from environmental degradation and inactivation. In planta use also avoids the time and expense of spraying or otherwise applying organisms and/or the toxin to the plant or the site of the pest in order to contact and control the target pest.
The subject B.t. toxin genes can be introduced via a suitable vector into a host, preferably a plant host. There are many compatible crops of interest, such as corn, cotton, and sunflowers.
Synthetic, plant-optimized genes, as exemplified herein, are particularly well suited for providing stable maintenance and expression of the gene in the transformed plant.
In some embodiments of the subject invention, transformed microbial hosts can be used in preliminary steps for preparing precursors that will eventually be used to transform plant cells and/or plants. Microbes transformed and used in this manner are within the scope of the subject invention. Recombinant microbes may be, for example, B.t., E. coli, or Pseudomonas (such as Pseudomons fluorescens). Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.
As an alternative to using plants transformed with a gene of the subj ect invention, the B.t. isolates, or recombinant microbes expressing the toxins described herein, can be used to control pests.
The B.t. isolates of the invention can be cultured using standard art media and fernentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores, crystals, and/or toxins can be formulated into wettable powders, liquid concentrates, granules, or other formulations by the addition of surfactants, dispersants, inert carriers and other components to facilitate handling and application for particular target pests. These formulation and application procedures are all well known in the art.
The subject invention also includes mutants of the above B.t. isolates which have substantially the same pesticidal properties as the parent B.t. isolates. Mutants can be made by procedures well known in the art. Ultraviolet light and nitrosoguanidine are used extensively toward this end. An asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate.
Suitable microbial hosts, e.g., Pseudomonas, transformed to express one or more genes of the subject invention can be applied to the situs of the pest, where the transformed host can proliferate and/or be ingested. The result is a control of the pest.
Alternatively, the microbe hosting the toxin gene can be killed and treated under conditions that prolong the activity of the toxin and stabilize the cell; the treated cell, which retains the toxic activity, then can be applied to the environment of the target pest. See, e.g., U.S. Pat. Nos. 4,695,462; 4,861,595; and 4,695,455. Thus, the invention includes the treatment of substantially intact B.t. cells, and/or recombinant cells containing the expressed toxins of the invention, treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest. Such treatment can be by chemical or physical means, or a combination of chemical or physical means, so long as the technique does not deleteriously affect the properties of the pesticide, nor diminish the cellular capability in protecting the pesticide. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes available to act as such upon ingestion by a target insect.
Synthetic Plant-optimized Genes
Preferred synthetic B.t. genes according to the present invention include nucleotide sequences that have: (1) more plant preferred codons than the native B.t. gene, (2) a frequency of codon usage that is closer to the codon frequency of the intended plant host than the native B.t. gene, or (3) substantially all codons comprised of the codon that has the highest frequency in the intended plant host. While the subject invention provides specific embodiments of synthetic genes that are particularly useful in transformed plants, other genes that are functionally equivalent to the genes exemplified herein can also be used to transform hosts, preferably plant hosts. Additional guidance for the production of synthetic genes for use in plants can be found in, for example, U.S. Pat. No. 5,380,831.
Polynucleotide Probes
One method for identifying useful toxins and genes is through the use of oligonucleotideprobes. These probes are detectable nucleotide sequences. Probes provide a rapid method for identifying toxin-encoding genes. The nucleotide segments which are used as probes can be synthesized using a DNA synthesizer and standard procedures.
It is well known that DNA possesses a findamental property called base complementarity. In nature, DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand. The base adenine (A) on one strand will always be opposed to the base thymine (T) on the other strand, and the base guanine (G) will be opposed to the base cytosine (C). The bases are held in apposition by their ability to hydrogen bond in this specific way. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining of the two complementary strands. These bonds can be broken by treatments such as high pH or high temperature, and these conditions result in the dissociation, or xe2x80x9cdenaturation,xe2x80x9d of the two strands. If the DNA is then placed under conditions which make hydrogen bonding of the bases thermodynamically favorable, the DNA strands will anneal, or xe2x80x9chybridize,xe2x80x9d and reform the original double stranded DNA. If carried out under appropriate conditions, this hybridization can be highly specific. That is, only strands with a high degree of base complementarity will be able to form stable double stranded structures. The relationship of the specificity of hybridization to reaction conditions is well known. Thus, hybridization may be used to test whether two pieces of DNA are complementary in their base sequences. It is this hybridization mechanism which facilitates the use of probes to readily detect and characterize DNA sequences of interest.
The probes may be RNA, DNA, or PNA (peptide nucleic acid). The probe will normally have at least about 10 bases, more usually at least about 17 bases, and may have up to about 100 bases or more. Longer probes can readily be utilized, and such probes can be, for example, several kilobases in length. The probe sequence is designed to be at least substantially complementaryto a portion of a gene encoding a toxin of interest. The probe need not have perfect complementarity to the sequence to which it hybridizes. The probes may be labelled utilizing techniques which are well known to those skilled in this art.
One approach for the use of probes entails first identifying by Southern blot analysis of a gene bank of the Bacillus isolate all DNA segments homologous with the disclosed nucleotide sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the probable activity of many new Bacillus isolates, and of the individual gene products expressed by a given Bacillus isolate. Such a probe analysis provides a rapid method for identifying potentially commercially valuable insecticidal toxin genes within the multifarious subspecies of B.t. The particular hybridization technique is not essential. As improvements are made in hybridization techniques, they can be readily applied.
One useful hybridizationprocedure typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically. Either lysed bacteria or total fractionated nucleic acid isolated from bacteria can be used. Cells can be treated using known techniques to liberate their DNA (and/or RNA). The DNA sample can be cut into pieces with an appropriate restriction enzyme. The pieces can be separated by size through electrophoresis in a gel, usually agarose or acrylamide. The pieces of interest can be transferred to an immobilizing membrane.
The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe""s detectable label provides a means for determining in a known manner whether hybridization has occurred.
In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include 32P, 35S, or the like. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or perixodases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probes can be made inherently fluorescent as described in International Application No. WO 93/16094.
Various degrees of stringency of hybridization can be employed, as described below. The more stringentthe conditions, the greaterthe complementaritythat is requiredfor duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.
Hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes can be performed by standard methods (Maniatis et al. [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). In general, hybridizationand subsequent washes can be carried out under low, moderate, and/or high stringency conditions that allow for detection of target sequences with homology to the exemplified toxin genes. For double-stranded DNA gene probes, hybridization can be carried out overnight at 20-25xc2x0 C. below the melting temperature (Tm) of the DNA hybrid in 6xc3x97 SSPE, 5xc3x97 Denhardt""s solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285).
Tm=81.5xc2x0 C.+16.6 Log[Na+]+0.41(%G+C)xe2x88x920.61(%formamide)xe2x88x92600/length of duplex in base pairs.
Washes are typically carried out as follows:
(1) Twice at room temperature for 15 minutes in 1xc3x97 SSPE, 0.1% SDS (low stringency wash).
(2) Once at Tmxe2x88x9220xc2x0 C. for 15 minutes in 0.2xc3x97 SSPE, 0.1% SDS (moderate stringency wash).
Other low stringency washes include 6xc3x97 SSPE, 0.1% SDS at 37xc2x0 C. or 2xc3x97 SSPE, 0.1% SDS at Tmxe2x88x9220xc2x0 C.
For oligonucleotide probes, hybridization can be carried out overnight at 10-20xc2x0 C. below the melting temperature (Tm) of the hybrid in 6xc3x97 SSPE, 5xc3x97 Denhardt""s solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes can be determined by the following formula:
Tm (xc2x0 C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs, S.V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown [ed.], Academic Press, New York, 23:683-693).
Washes are typically carried out as follows:
(1) Twice at room temperature for 15 minutes 1xc3x97 SSPE, 0.1% SDS (low stringency wash). (2) Once at the hybridizationtemperaturefor 15 minutes in 1xc3x97 SSPE, 0.1% SDS (moderate stringency wash).
In general, salt and/or temperature can be altered to change stringency. In addition, formamide or aqueous washes can be used. Formamide washes require a lower temperature than aqueous washes. With a labeled DNA fragment  greater than 70 or so bases in length, the following conditions (aqueous washes) can be used:
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, useful probe sequences can include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan; other methods may become known in the future. These variants can be used in the same manner as the original primer sequences so long as the variants have substantial sequence homology with the original sequence. As used herein, substantial sequence homology refers to homology which is sufficient to enable the variant probe to function in the same capacity as the original probe. Preferably, this is greater than 50%; more preferably, this homology is greater than 75%; and most preferably, this homology is greater than 90%. The degree of homology needed for the variant to function in its intended capacity will depend upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations which are designed to improve the function of the sequence or otherwise provide a methodological advantage.
PCR Technology
Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202,and 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim [1985] xe2x80x9cEnzymatic Amplification of xcex2-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia,xe2x80x9d Science 230:1350-1354.). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3xe2x80x2 ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5xe2x80x2 ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.
DNA sequences can be designed and used as primers for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5xe2x80x2 end) can be produced in a given primer by methods known to an ordinarily skilled artisan.
All of the references cited herein are hereby incorporated by reference.